The number of control systems useful for expressing heterologous genes in E. coli is somewhat limited. While it is difficult to predict or develop a system that will assuredly lead to high level expression of any targeted protein, there are a number of desirable characteristics that should be taken into account when designing an expression system. First, the promoter chosen to regulate heterologous gene expression should be easily and inexpensively induced in fermentation conditions. Second, an expression system should be tightly regulated since high level expression of a protein in E. coli may be detrimental or lethal to the cell. In addition, expression vectors should be designed with multiple cloning sites to facilitate subcloning of foreign genes and gene fragments.
High level expression of heterologous genes in bacteria can be achieved by using a tightly regulatable promoter such as the E. coli lac promoter (see, e.g. Backman, K. and M. Ptashne, (1978) "Maximizing gene expression on a plasmid using recombination in vitro." Cell 13:65-71); the trp promoter (see, e.g. Nichols, B. P. and C. Yanofsky, (1983) "Plasmids Containing the trp Promoters of Escherichia coli and Serratia marcescens and Their Use in Expressing Cloned Genes." Methods in Enzym. 101:155-164); the .lambda. phage L promoter (See e.g. Rosenberg, M., et al. (1983) "The use of pKC30 and its Derivatives for Controlled Expression of Genes." Methods in Enzym. 101:123-138); and the T7 .phi.10 promoter (See e.g. Studier, F. W. and B. A. Moffatt (1986). "Use of Bacteriophage T7 RNA Polymerase to Direct Selective High-level Expression of Cloned Genes." J. Mol. Biol. 189:113-130). All of these promoters, except for the T7 .phi.10, are regulated by the presence or absence of their cognate repressor. Maintaining repression of these promoters when they are on multicopy or runaway plasmid replicons can be difficult. As a consequence, expensive or awkward procedures must be used to inactivate the repressor in these negatively regulated systems.
The T7 .phi.10 promoter is activated in a positive fashion by the presence of the T7 polymerase. In the absence of the T7 polymerase, transcription from .phi.10 promoters is very low. Since the T7 polymerase gene itself is located in the E. coli chromosome, in single copy and controlled by the lac repressor, its synthesis can be well controlled. This arrangement has the advantage of keeping the promoter inactive regardless of the promotor copy number, resulting in tighter regulation of the expression system.
The phage P2 contains four late operons encoding packaging and lysis genes. (See, e.g., Lindahi, G. (1971) "On the Control of Transcriptions in Bacteriophage P2." Virol. 46(3):620-633; Sunshine, M. G., et al. (1971) "P2 Phage Amber Mutants: Characterization by Use of a Polarity Suppressor." Virol: 46(3):691-702; and Geisselsolder, J. M., et at. (1973): "In Vivo Transcription Patterns of Template Coliphage P2" J. Mol. Biol. 77(3):425-415). Transcription of these operons, F,O,P and V, requires both E. coli RNA polymerase and the product of the P2 ogr gene (See, e.g. Lindqvist, B. H. (1974) "Expression of Phage Transcription in P2 Lysogens Infected with Helper-dependent Coliphage P4" Proc. Natl. Acad. Sci. USA 71(7):2770-2774; Sunshine, M. G. and B. Sauer, (1975) "A Bacterial Mutation Blocking P2 Phage Late Gene Expression." Proc. Natl. Acad. Sci. USA 72(7):2770-2774). Examination of the sequences of these late promoters has revealed the absence of a strong consensus E. coli promoter (-35 and -10 regions).
P4, a satellite virus of bacteriophage P2, does not encode its own morphogenic functions but can be supplied with them by a co-infecting P2 virion. (See e.g., Six, E. W. (1975) "The Helper Dependence of Satellite Bacteriophage P4: Which Gene Functions of Bacteriophage P2 are Needed by P4?" Virol.67:249-26). One of the late P4 proteins, .delta., can transactivate the P2 late promoters in a manner analogous to the P2 ogr protein. (See, e.g., Souza, L. (1977) "A Transactivation Mutant of Satellite Phage P4." Virol. 81:81-90). In addition, the .delta. protein can activate one of P4's own promoters (psid), which in turn encodes the delta gene (See, e.g. Dale, E. C. et al. (1986) "Organization and Expression of the Satellite Bacteriophage P4 Late Gene Cluster," J. Mol. Biol. 192:793-803).
The instant invention provides a novel tightly regulated high-level heterologous gene expression system for E. coil based on the principle of positive activation of a P2 F promoter by the regulatable expression of the P4 delta gene. The P2 F promoter, which is synthesized beginning at position -70, with respect to the start site of transcription, is cloned into a multicopy plasmid. E. coli RNA polymerase is activated to transcribe the F promoter by the P4 .delta. protein which is supplied, in a sufficient quantity, by a single or a few copies of the delta gene which is under control of a regulatable promoter in a lysogenic host. Accordingly, the present invention provides an inexpensive and tightly regulatable expression system useful to produce heterologous proteins in E. coli hosts.